Method of regulating immune function

ABSTRACT

Disclosed herein is a method of treating an immune system dysfunction in a mammal by administering a prolactin reducer and a prolactin enhancer at a predetermined time or times.

BACKGROUND OF THE INVENTION

This is a continuation of application Ser. No. 08/780,727 filed Jan. 8,1997 (now U.S. Pat. No. 5,872,127) which is a continuation ofapplication Ser. No. 08/271,881 filed Jul. 7, 1994 (now U.S. Pat. No.5,696,128).

FIELD OF THE INVENTION

This invention relates to methods for rectifying or amelioratingabnormal responses of the mammalian immune system, and modifying normalresponses of the mammalian immune system. More particularly, thisinvention relates to methods employing the alteration of prolactinrhythms as a method of adjusting mammalian immune response.

PROLACTIN AND IMMUNITY

The importance of neuroendocrine regulation of immunity has becomeincreasingly evident during the past decade (Besedovsky, H. O. et al.,J. Immunol. 135:750s-754s, 1985; Blalock, J. E., Physiol. Rev. 69: 1-54,1989; Berozi, I., Dev. Comp. Immunol. 13:329-341, 1989). Much of thisinterest has focused on the anterior pituitary hormone prolactin, whichhas been reported to have potent, albeit inconsistent and oftenconflicting, effects on immune activity (Gala, R. R., Proc. Soc. Exp.Biol. Med. 198:5-13, 1991; Nicoletti, J. et al., Reprod. Immunol.15:113-121, 1989; Vidaller, A., et al., Clin. Immunol. Immunopathol.38:337-343, 1986; Gerli, R. et al., Clin. Immunol. 7:463-470, 1987).

The role of prolactin in immunity is exemplified by studiesdemonstrating exogenous prolactin-induced restoration of immunecompetence in hypophysectomized mammals (Gala, R. R., Proc. Soc. Exp.Biol. Med. 198:5-13, 1991; Bercal, I. et al., Acta Endocrinol.98:506-513, 1981). In intact animals, prolactin administration has beenassociated with numerous immunological effects including stimulation ofcellular or antibody responses, as well as stimulation of various immunesystem upregulating substances such as IL-2 (both IL-2 production andIL-2 receptor expression); enhancement of lymphocyte number, activityand mitogenic responses; and augmentation of macrophage cytotoxicity(Gala, R. R., Proc. Soc. Exp. Biol. Med. 198:5-13, 1991; Bernton, E. W.et al., Science 239:401-404, 1988; Rovensky, J. et al., Int. J. Immuno.Pharmac. 13:267, 1991.)

Other lines of evidence reveal an association between hyperprolactinemia(i.e. elevated levels of circulating endogenous prolactin) which is dueto natural, pathological, pharmaceutical, or stress conditions, andstates of immune dysfunction, such as immunosuppression or autoimmunediseases. The autoimmune diseases for which exacerbative associationswith prolactin have been observed in the past include rheumatoidarthritis, systemic lupus erythematosus (SLE) and multiple sclerosis.Nicoletti, J. et al., Reprod. Immunol. 15:113-121, 1989; Vidaller, A.,et al., Clin. Immunol. Immunopathol. 38:337-343, 1986; Gerli, R. et al.,Clin. Immunol. 7:463-470, 1987; McMurray, R. et al., J. Immunol.147:3780, 1991.

In light of these apparently conflicting results, (increased prolactinlevel-associated augmentation of allo-immune response, exacerbatedauto-immune response, and immuno-suppression) the effects of elevatedblood prolactin levels on the immune system have been far from clear.

In recent years, research has focused on improving the ability of theimmune system to combat various diseases including malignancies.Experimental evidence that major histocompatibility antigens have animportant role in host defenses against the development and spread oftumors has been rapidly accumulating.

Another line of research has specifically focused on suppression ofautoimmune diseases, which are characterized by the inability of theimmune system to recognize self tissue as "self" and by the mounting ofan immune response against self tissue as though it were a foreignantigenic substance.

Yet another area of intensive immunological research is focused onvarious immunodeficiencies including AIDS. Despite intense researchhowever, progress is slow and the immune mechanisms involved are provingelusive.

Numerous potential immunomodulatory agents are under currentinvestigation by third parties for clinical usefulness. These agentsinclude biologically derived compounds such as interferons andinterleukins (and synthetic compounds such as isoprinosine andpyrimidinones). Although interferons and other cytokines and lymphokinesare naturally occurring substances, their clinical use (which hasinvolved administration by injection) has not been consistentlybeneficial (and/or the favorable results have been short-lived).Furthermore, cytokine and lymphokine therapies are most oftenaccompanied by severe side effects such as toxicity and fever.

Accordingly, there is a need in the field of immunology for agents whichmodify pathological immune system responsiveness and regulate theendogenous production of substances which are in turn native immunesystem regulators. Use of such agents to "re-program" the immune system:(i) would improve host resistance to infection and ability to combatexisting infections; (ii) overcome immunosuppression, abateimmunodeficiency, and improve immunity against tumors and restore normalimmune function; and (iii) prevent or suppress autoimmunity and restorenormal immune function.

PROLACTIN AND CIRCADIAN RHYTHMS

Research has demonstrated that circadian rhythms play important roles inregulating prolactin activities and vice versa.

Publications such as Meier, A. H., Gen. Comp. Endocrinol. 3(Suppl1):488-508, 1972; Meier, A. H., Trans. Am. Fish. Soc. 113:422-431, 1984;Meier, A. H. et al., Current Ornithology II (ed Johnston R. E.) 303-343,1984; Cincotta, A. H. et al., J. Endocrinol. 120:385-391, 1989; Meier,A. H., Amer. Zool. 15:905-916, 1975; Meier, A. H., Hormonal Correlatesof Behavior (eds. Eleftherton and Sprott) 469-549, 1975 illustrate howcircadian rhythms regulate prolactin activities. The resulting dailyvariations in responsiveness of various cell types to prolactin have aprimary role in regulating numerous physiological processes, includingfat storage, lipogenic responsiveness to insulin, migratory behavior,metamorphosis, reproduction, growth, pigeon cropsac development andmammary development (Meier, A. H., Gen. Comp. Endocrinol. 3(Suppl1):488-508, 1972; Meier, A. H., Amer. Zool. 15:905-916, 1975; Meier, A.H. et al., Science 173:1240-1242, 1971). In regulating one of theforegoing physiological activities, prolactin may be observed to producea stimulatory or an inhibitory effect on a given activity, or to have noeffect on it. These varying effects have recently been shown in animalsto be a function of the time of the daily endogenous peak (i.e.acrophase) of the rhythm of plasma prolactin concentration or a functionof the time of daily injection of exogenous hormone (or of a substancethat increases prolactin levels) or of the relation between endogenouspeak and any induced peak. Furthermore, high levels of prolactinrestricted to a discreet daily interval have a much greater physiologic(e.g. metabolic) effect in animals than do constant high levelsthroughout a day (Cincotta, A. H. et al., Horm. Metab. Res. 21:64-68,1989; Borer, K. T. in The Hamster: Reproduction and Behavior (ed.Siegel, H. I.) 363-408, 1985). Such findings demonstrate the existenceof daily response rhythms to prolactin by certain types of cells.

The first demonstration of a daily variation in physiologicalresponsiveness to any hormone was the dramatic variation in fatteningresponsiveness to prolactin in the white-throated sparrow (Meier, A. H.et al., Gen. Comp. Endocrinol. 8:110-114, 1967). Injections at midday ofa 16-hour daily photoperiod stimulated 3-fold increases in body fatlevels, whereas injections given early in the photoperiod reduced fatstores by 50%. Such daily variations in fattening responses to prolactinwere subsequently demonstrated in numerous species of all the majorvertebrate classes (Meier, A. H., Amer. Zool. 15:905-916, 1975; Meier,A. H., Hormonal Correlates of Behavior (eds. Eleftherton and Sprott)469-549, 1975) indicating the fundamental nature of such a temporalorganization. The fattening response rhythm persists under constantlight conditions (Meier, A. H. et al., Proc. Soc. Exp. Biol. Med.137:408-415, 1971) indicating that it, like many other endogenous dailyvariations, is a circadian rhythm.

Additional studies have demonstrated that circadian rhythms have primaryroles in regulating numerous physiologic activities such as, lipidmetabolism and body fat stores (Meier, A. H. et al., Current OrnithologyII (ed Johnston R. E.) 303-343, 1984; Meier, A. H., Amer. Zool.15:905-916, 1975; Meier, A. H., Hormonal Correlates of Behavior (eds.Eleftherton and Sprott) 469-549, 1975; Meier, A. H. et al., J. Am. Zool.16:649-659, 1976); Cincotta et al., Life Sciences 45:2247-2254, 1989;Cincotta et al., Ann. Nutr. Metab. 33:305-14, 1989; and Cincotta et al.,Horm. Metabol. Res. 21:64-68, 1989. These experiments showed that aninteraction of circadian rhythms of liporegulatory hormones (stimuli)and of circadian responses to these hormones (in target cells)determines amount of lipogenesis and fat storage. Thus, high plasmaconcentrations of prolactin (which serves as the stimulus) occur duringthe daily interval of maximal fattening responsiveness to prolactin infat animals, but occur at other unresponsive times of day in leananimals (Meier, A. H., Amer. Zool. 15:905-916, 1975; Meier, A. H.,Hormonal Correlates of Behavior (eds. Eleftherton and Sprott) 469-549,1975; Speiler, R. E. et al., Nature 271:469-471, 1978). Similarly,plasma insulin (which acts as the stimulus) levels are highest duringthe daily interval of greatest hepatic lipogenic response to insulin inobese hamsters, but at a different time of day in lean hamsters(deSouza, C. J. et al., Chronobiol. Int. 4:141-151, 1987; Cincotta, A.H. et al., J. Endocr. 103:141-146, 1984). The phase relationships ofthese stimulus and response rhythms are believed to be expressions ofneural circadian centers which in turn can be reset by neurotransmitteragents and hormone injections (including prolactin) to produce eitherfat or lean animals (Meier, A. H., Trans. Am. Fish. Soc. 113:422-431,1984; Meier, A. H. et al., Current Ornithology II (ed Johnston R. E.)303-343, 1984; Cincotta, A. H. et al., J. Endocrinol. 120:385-391, 1989;Emata, A. C. et al., J. Exp. Zool. 233:29-34, 1985; Cincotta, A. H. etal., Chronobiol. Int'l 10:244-258, 1993; Miller, L. J. et al., J.Interdisc. Cycles Res. 14:85-94, 1983). Accordingly, timed prolactinadministration or enhancement acts directly upon tissues (e.g. liver inlipogenesis) undergoing circadian rhythms of responsiveness to thehormone to produce daily variations in net physiologic effects(Cincotta, A. H. et al., Horm. Metab. Res. 21:64-68, 1989) and actsindirectly by resetting one of the circadian neuroendocrine oscillationsof a multi-oscillatory circadian pacemaker system to establish differentphase relations between the multiple circadian (neural, hormonal, andtissue) expressions that control lipid metabolism (Meier, A. H., Trans.Am. Fish. Soc. 113:422-431, 1984; Meier, A. H. et al., CurrentOrnitholoay II (ed Johnston R. E.) 303-343, 1984; Cincotta, A. H. etal., J. Endocrinol. 120:385-391, 1989; Emata, A. C. et al., J. Exp.Zool. 233:29-34, 1985; Cincotta, A. H. et al., Chronobiol. Int'l10:244-258, 1993; Miller, L. J. et al., J. Interdisc. Cycles Res.14:85-94, 1983).

The present inventors have previously shown that prolactin, orsubstances that affect circulating prolactin levels, also affectcircadian rhythms and in fact can be used to modify such rhythms (sothat they more closely resemble the rhythms of lean, healthy, youngindividuals of the same sex) and to reset such rhythms (so that theypersist in the modified condition). See, e.g. U.S. patent applicationsSer. Nos. 08/158,153 [07850], 07/995,292 [07788], 07/719,745 [17849],07/999,685 [07848] and 08/171,569. This prior work by the presentinventors has been clinically tested in humans afflicted with variousmetabolic disorders (obesity, diabetes and others) with very favorableresults.

In particular, in U.S. patent application Ser. No. 07/995,292, and inits continuation-in-part Ser. No. 08/264,558, filed Jun. 23, 1994, thepresent inventors disclose a method for the reduction in a subject,vertebrate animal or human, of body fat stores, and reduction of atleast one of insulin resistance, hyperinsulinemia, and hyperglycemia,and other metabolic diseases, especially those associated with Type IIdiabetes. More specifically, the foregoing application discloses methodsfor: (i) assessing the daily prolactin level cycles of a normal(healthy) human or vertebrate animal (free of obesity, disease or otherdisorder); (ii) diagnosing aberrant daily prolactin level cycles of ahuman or vertebrate animal; and (iii) determining the appropriateadjustments that need to be made to normalize such aberrant prolactinlevel cycles. This method involves the administration of at least one ofa prolactin reducer and/or a prolactin enhancer at a first predeterminedtime (or times) within a 24-hour period (if only a prolactin reducer isadministered) and/or at a second predetermined time (or times) of a24-hour period (if a prolactin enhancer is administered). This therapy,when continued for several days, weeks or months, results in thelong-term adjustment of aberrant or abnormal prolactin level cycles sothat they conform to (or simulate) normal prolactin level cycles. Thisbenefit persists over the long-term even after cessation of therapy. Asa result, aberrant physiological parameters associated with variousmetabolic disorders are restored to normal levels or are modified toapproach normal levels. Although this method is applied to all personshaving aberrant prolactin levels during at least a portion of a 24-hourperiod, it does not mention the possibility of applying it to personssuffering from immune dysfunction.

Thus, the mutual dependence of prolactin and circadian rhythms andparticularly the time-sensitivity of such dependence has not previouslybeen correlated with immune function or dysfunction. The presentinventors postulated (i) a similar daily variation of the response ofthe immune system to prolactin and (ii) an ability of timed, inducedvariations in prolactin levels to modulate immune responses byinfluencing production of naturally occurring immune system (up- ordown-) regulators. Experimental confirmation of these postulates gaverise to the present invention, and resolved the apparent conflicts inthe effects of prolactin on immunity.

SUMMARY OF THE INVENTION

One aspect of the present invention is directed to a method ofameliorating or rectifying immune system abnormalities in a mammal inneed of such treatment. The method involves the administration to themammal of a prolactin reducer and/or enhancer at a predetermined time ortimes during a 24-hour period that results in modification of themammal's abnormal prolactin profile so that it approaches or conforms tothe prolactin profile of a young healthy mammal of the same species.

Another aspect of the present invention is directed to a method ofameliorating or rectifying immune system abnormalities on a long-termbasis by continuing the foregoing timed administration(s) of theprolactin reducer and/or enhancer until the altered prolactin rhythm ofthe subject is reset and persists in this reset condition for anextended period of time even after cessation of therapy, resulting inpersistence of the improvement of immune system abnormalities.

Yet another aspect of the invention is directed to a method ofaugmenting (upregulating) immune response in a mammal (e.g., for thepurpose of increasing the subject's ability to mount an immune responseagainst infection). The method involves the timed administration of aprolactin reducer and/or enhancer at a time or times (respectively) atwhich reducing (or enhancing) the subject's plasma prolactin levelswould enhance the subject's ability to mount an immune response. Thismethod may also be practiced on subjects having a normal immune system.

Thus, the present invention is directed to adjusting the phaserelationship between the circadian rhythms for prolactin and for one ormore immune responses. The invention involves normalizing (or resetting)the circadian rhythm for prolactin to resemble that of a healthy youngsubject. The invention also involves manipulating the circadian rhythmfor prolactin to bring it in such a phase and amplitude relation withthe immunologic responsiveness to prolactin as to exert an amplifyingeffect on a predetermined aspect of the immune response.

"Immune dysfunction" or "immune abnormality" means individually orcollectively a state of immunodeficiency or immunosuppression (marked byinability or compromised ability to mount an immune response against apathogen or other affliction such as a tumor) and/or a state ofmistargeted immune activity such as autoimmunity. Immunodeficiency andimmunosuppression include situations where a subject has reduced abilityto mount a T-cell response or a B-cell response (as evidenced forexample by reduced mixed lymphocyte reaction, reduced delayed-typehypersensitivity or reduced T- or B-cell proliferation to a stimulus);or has reduced ability to produce cytokines or lymphokines orantibodies; or exhibits reduced expression of lymphokine receptors orreduced antigen-presenting ability (as evidenced for example by reducedexpression of Class I or Class II Major Histocompatibility Complex).Such compromised ability to mount an immune response can be the resultof congenital or acquired immunodeficiency or the result of chemotherapyor radiation, or other drug-induced immunosuppression. Consequently, arectification or amelioration of immune dysfunction is the total orpartial restoration of one or more of the foregoing immune responses.

"Prolactin reducer" is a substance or composition that has the abilityto lower circulating prolactin levels upon administration to a mammal;"prolactin enhancer" is a substance or composition that has the abilityto raise circulating prolactin levels, and includes prolactin itself.

Prolactin reducers and prolactin enhancers are referred to collectivelyas "prolactin modulators".

"Prolactin profile" of a subject is a depiction of circulating prolactinlevels and their variation over all or part of a 24-hour period, andtherefore an expression of all or part of the subject's plasma prolactindaily rhythm.

"Healthy" is a young, lean subject free of disease includingmalignancies, immune system dysfunctions and metabolic abnormalities. Ahealthy subject is one with a normal prolactin profile, i.e., aprolactin profile that does not depart from the baseline of thatsubject's species and sex by more than one standard error of the mean(SEM). The normal or baseline profile for healthy male and female humansis depicted in FIG. 1.

In order to avoid "false positives" a subject will not generally beconsidered to have an abnormal prolactin profile unless:

(a) the subject's daytime blood prolactin level is at least 1 SEM higherthan the baseline at two (or more) time points during daytime spacedapart by at least one and preferably by at least two hours; or

(b) the subject's daytime blood prolactin level is at least 2 SEM higherthan the baseline at one time point during daytime; or

(c) the subject's night time blood prolactin level is at least 1 SEMbelow the base line at two (or more) spaced apart time points (as in(a)); or

(d) the subject night time blood prolactin level is at least 2 SEM belowthe base line at one time point during night time.

The human male and female baselines are depicted in FIG. 1. One SEMduring waking hours (07:00-22:00) is about 1-2 ng/ml for males and about1-3 ng/ml for females; one SEM during night time (22:00-07:00) is about3 ng/ml for males and about 3-6 ng/ml for females.

The characteristics of the prolactin level daily rhythm or profile thatare to be approached or conformed in humans include achieving lowprolactin levels (2-7 ng/ml of plasma) for males and 2-10 ng/ml forfemales) during most or all of the time period between 07:00 and 22:00h.

Ideally, a peak prolactin level should also be achieved between thehours of 22:00 and 07:00 (preferably between 1:00 and 4:00) (the peakshould be at least 10 ng/ml and most preferably between 10-15 ng/ml formales and at least 15 ng/ml and preferably between 15 and 25 ng/ml forfemales).

Advantages of the present invention include:

upregulation of immune responses when needed to combat disease;

restoration of normal immune responses (abatement of autoimmunity,immunodeficiency).

The benefits of the present invention may persist long-term even aftercessation of the administration of prolactin modulators.

Other features and advantages of the present invention will be apparentfrom the following description taken in conjunction with theaccompanying drawings.

BRIEF DESCRIPTION OF THE DRAWINGS

FIG. 1 is the baseline prolactin daily rhythm or profile curve forhealthy males and females.

FIGS. 2 and 3 are bar diagrams showing the relationship between mixedlymphocyte reaction (MLR) and time of day of prolactin administration.An asterisk denotes a significant difference from control (p<0.05;Student's t test)

FIG. 4 is the same type of diagram as FIG. 2 but showing therelationship between MLR and time of day of administration of theprolactin-enhancer domperidone.

FIG. 5 is the same type of diagram as FIG. 4 but the prolactin enhanceris 5HTP.

FIGS. 6A and 6B are the same type of diagrams as FIG. 3 but showing therelationship between MLR and time of day of a prolactin reduceradministration; FIG. 6A: 200 μg bromocriptine; FIG. 6B: 50 μgbromocriptine.

FIG. 7 is a bar diagram showing the relationship between T-cell responseto the stimulus Concanavallin A (ConA) and the time of bromocriptineadministration.

FIG. 8 is the same type of diagram as FIG. 7 but for B-cell response tothe stimulus lipopolysaccharide (LPS).

FIG. 9 is a bar diagram showing the relationship between delayed-typehypersensitivity (DTH) responses (foot pad swelling) and time of day ofprolactin administration.

FIG. 10 is the same type of diagram as FIG. 9 but represents the meanpercent inhibition of foot pad swelling compared to the positivecontrols obtained from 4 experiments. An asterisk denotes a significantdifference from the positive control in millimeters of foot pad swelling(p<0.008; Student's t test).

FIG. 11 is a bar diagram showing the relationship between thymus cellnumber and time of day of prolactin administration in treated andcontrol mice. The results represent the mean cell number +/- SEM of 8-10mice per group. An asterisk denotes a significant difference fromcontrol (p<0.01; Student's t test).

FIG. 12 is a series of tracings depicting the male base prolactinprofile (i.e the normal prolactin profile for healthy young males), and,superimposed on it, prolactin level profiles (ng/ml plasma) pre-therapyand in-therapy (visit 2 and visit 3) prolactin profiles for a malepatient suffering from Crohn's disease.

FIGS. 13 and 14, respectively contain the female base prolactin profileand tracings similar to those of FIG. 12 for two female rheumatoidarthritis patients.

FIGS. 15 and 16, respectively contain the female base prolactin profileand tracings similar to those of FIG. 12 for two female fibromyalgiapatients.

DETAILED DESCRIPTION OF THE INVENTION

All patents, patent applications, and literature references discussed inthis specification are hereby incorporated by reference. In case ofconflict, the present disclosure controls.

Effects of Prolactin Modulation on Immune Responses

The alteration of prolactin levels in a subject having a normal immunesystem (either by administering prolactin, or by administeringsubstances that alter prolactin blood levels) has been found to augmentor reduce a subject's ability to mount an immune response to a givenchallenge. Whether the effect on the immune response is stimulatory orsuppressive is dependent on the time of day the alteration of theprolactin levels occurs and on the nature of the alteration. Thus,increasing the plasma levels of the hormone at or near a time whencellular responsiveness to high prolactin is at its peak, in micepreferably about 10-12 hours after light onset (HALO), normal immuneresponses (and immune responses to alloantigens) are augmented.Conversely, decreasing prolactin plasma levels at or near the peak ofresponsiveness, in mice 4-12 HALO, preferably 10-12 HALO, suppressesimmune response. On the other hand, causing the circulating prolactinlevels to increase at a time when cellular responsiveness to prolactinis at its lowest, in mice preferably at approximately light onset (20-24HALO and 0-3 HALO; preferably 22-24 HALO and 0-2 HALO), immune responsesare often (but not always) suppressed.

The experimental data described herein show that prolactin injections(or prolactin enhancer administration) 9-12 HALO cause an increase inthe mouse mixed lymphocyte response (MLR) to alloantigens and anincrease in the proliferation of nonstimulated mouse splenocytes ascompared to naive controls. Prolactin injections (or prolactin enhanceradministration) made 16-24 HALO did not have a significant effect onMLR. Prolactin injections (or enhancer administration) at light onsetresulted in significant inhibition of mouse immune responsiveness (asmeasured by MLR) as compared to naive controls. These results indicatethat the effect of in vivo prolactin modulation of in vitro immuneresponses to foreign antigen is time-of-day dependent. In vivo responseto antigen as measured by delayed-type hypersensitivity (DTH)experiments is also described herein. As with the MLR above, prolactininjections made at light onset often (but not always) inhibited the footpad swelling response, indicating that prolactin caused a reduced immuneresponse; however, prolactin administration at 10 HALO was significantlystimulatory relative to control.

A time of day dependent role for prolactin in immune responses is alsoindicated by results of experiments on mice which decrease prolactinblood levels (by administration of a prolactin reducer) during specificdaily intervals of daily immune responsiveness to exogenous prolactin(i.e. during an interval about 9-12 HALO in mice and another intervalabout 0 HALO in mice). Dose-response studies with bromocriptine, a D2dopamine agonist which inhibits endogenous prolactin secretion, indicatethat bromocriptine exerted an inhibitory action on the DTH response at10 HALO but not at 0 HALO. Bromocriptine was also found to be inhibitoryfor T and B cell proliferative responses to mitogenic stimulation witheither concanavalin A (100% ; p<0.01) or lipopolysaccharide (47% ;p<0.01) respectively, when administered at 10 but not at 0 HALO.

The above in vitro and in vivo immune responses are dependent on matureT cell activation. Thymic hormones are essential for the differentiationof progenitor T cells within the thymus. Moreover, thymic hormonesenhance peripheral T cell activity (Baxevanis, C. N. et al., Immunopharm15:73-84, 1988), major histocompatibility complex class II antigenexpression (Baxevanis, C. N. et al., J. Immunol. 148: 1979-1984, 1992),and augment antigen presenting function (Tzehoval, E. et al.,Immunopharm. 18:107-113, 1989), all of which can promote MLR and DTHreactivity. Inasmuch as prolactin stimulates thymic epithelial cellproliferation as well as thymic hormone production (Dardenne, M. et al.,Endocrinology 125:3-12, 1989), prolactin should also have an effect onthymus cell number. Indeed, daily prolactin injections were given to 5week old mice either at light onset or at 11 HALO for one month.Prolactin treatment at 11 HALO significantly increased thymus cellnumber relative to controls whereas prolactin injections at light onsetdid not.

The above results indicate the immunomodulatory effects of prolactinlevels and the relationship of cellular responsiveness to exogenousprolactin (or prolactin enhancers or reducers), and the time of day ofprolactin reduction or enhancement.

Although the foregoing experiments were conducted in mice, they aredependent on features of the immune system that are common to mammalshaving a prolactin daily rhythm including humans. These results showthat the blood levels of prolactin can be manipulated duringpredetermined intervals to bring about a desirable effect on the immunesystem.

According to the method of the present invention, the alteration ofprolactin levels of a subject at particular times of day providesmethods of improving immune responsiveness of the subject or restoringor augmenting normal immune responses or ameliorating abnormal immuneresponses. The method may be used to increase the protection of subjectsthat are immunosuppressed (or even subjects that do not suffer fromimmunosuppression) against infection. Augmenting the immune responsewill provide an increased level of protection against invading pathogenssuch as viruses, bacterial, or fungal infections in susceptibleindividuals. This method will also be useful in the treatment ofindividuals who are immunocompromised or immunodeficient independent ofthe cause. Additional subjects who could benefit from this treatmentmethod include without limitation allograft recipients, surgerypatients, allergy sufferers, burn victims, cancer patients receivingchemotherapy or radiation therapy, patients suffering from HIV-infectionor a congenital immunodeficiency such as severe combinedimmunodeficiency (SCID) or DiGeorge Syndrome. Any subject whose immunesystem has been deregulated (but not completely ablated) by a congenitalor clinical condition or by medication will benefit from the presentinvention. An augmentation in immune responses is also of value ingroups sharing common quarters, such as military recruits, summercampers, or disaster victims, or with the aged in nursing homes, who areat a high risk of contracting infections.

The method can also be used to reduce or eliminate damage to a subjectcaused by a deleterious immune reaction. Specifically, subjectssuffering from autoimmune diseases whether such conditions are mediatedor dependent on B-cells, T-cells or both. Nonlimiting examples includerheumatoid arthritis, multiple sclerosis, endocrine ophthalmopathy,uveoretinitis, the autoimmune phase of Type 1 diabetes, systemic lupuserythematosus, myasthenia gravis, Grave's disease, glomerulonephritis,autoimmune hepatological disorder, autoimmune inflammatory boweldisease, and Crohn's disease. Subjects suffering from inflammationhaving immune reaction characteristics (e.g. anaphylaxis, allergicreaction) would also benefit from the present treatment method. Thismethod is also useful in the treatment of recipients of tissue or organtransplants to reduce host-induced allograft rejection.

Use of Prolactin Modulators to Alter Immune Response

(a) Adjusting Prolactin Rhythms of Subjects With Immune Dysfunction

It is known that young adult healthy mammals of a given species (andsex), e.g. humans (suffering from no hormonal or metabolic disorders orcancer or other infection or ailment) have highly predictable dailyprolactin level rhythms or profiles. The baseline curve for healthyhuman males and females in FIG. 1 is derived from such young healthyindividuals.

It is also known that persons suffering from immune dysfunction haveabnormal prolactin rhythms. Nicoletti, supra; Vidaller, supra; Gerli,supra; McMurray, supra, Fraga, A. et al., Arthritis Rheum. 32:524, 1989;and Laualle, C., J. Rheumatol. 14:266, 1987.

The phase relationship between the daily peaks of the stimulus (plasmaprolactin) rhythm and response (immunocellular rhythm) to prolactin isof critical importance to the status of immune function. Environmentaland pharmaceutical factors influencing either of these rhythms can beexpected to impact immune function. Furthermore, phase shifts in eitheror both of these rhythms may be associated with immunologic disorders,as well as cancer (Bartsch, C. et al., J. Pineal Res. 2:121-132, 1985;Bartsch, C. et al., Cancer 64:426-433, 1989).

For example, persons with autoimmune disease commonly havehyperprolactinemia during the day, especially in AM after dawn at whichtime, in humans, it is believed that the excess (above baseline)prolactin deregulates immune function. By adjusting (reducing) thedaytime prolactin levels of such individuals the deregulation of immunefunction can be rectified or ameliorated. In terms of the foregoingexperiments this would be equivalent to an animal the immune function ofwhich has been deregulated by administration of prolactin, e.g. at zeroHALO. The immune function can be restored by administration of aprolactin reducer at zero HALO.

Persons with immune dysfunction thus benefit to a significant extent byadjustment of their prolactin daily rhythms (as expressed by theirprolactin profile) to conform to or approach the normal or baselineprolactin curve of FIG. 1. An adjusted profile approaches a normal orhealthy profile, if all or a portion of the abnormal profile moves inthe correct direction by at least 2 ng/ml.

This adjustment can be accomplished by administration to suchindividuals of one or both of the following:

a prolactin reducer at a first predetermined time (or at more than onefirst predetermined time) and in a first amount effective to reduce daytime prolactin levels if these levels are too high; and

a prolactin enhancer at a second predetermined time (or at more than onesecond predetermined times) and in a second amount effective to increasenight time prolactin levels if these levels are too low.

In general, if a prolactin level altering substance is to beadministered, appropriate allowance should be made with respect to thetime of administration to permit that substance (depending on itspharmacokinetic properties) to affect prolactin levels such thatprolactin levels would be modified during the appropriate time of day.Thus, the prolactin altering substance will be administered as follows:

(a) if prolactin is administered, it will be administered during thetime interval that prolactin levels need to be raised;

(b) if a prolactin enhancer other than prolactin is administered, itwill be administered during or slightly prior to the time interval whenprolactin levels need to be raised (how much prior depends onpharmacokinetic properties: generally 0-3 hours prior will beeffective); and

(c) if a prolactin reducer is administered it will also be administeredduring or slightly prior to the time that prolactin levels need to bereduced (again, 0-3 hours prior will be generally effective).

In the method of the present invention, "prolactin enhancer" includesprolactin as well as substances which increase circulating prolactinlevels (e.g. by stimulating prolactin secretion). Non-limiting examplesof a prolactin enhancer include prolactin; melatonin; dopamineantagonists such as metoclopramide, haloperidol, pimozide,phenothiazine, domperidone, sulpiride and chlorpromazine; serotoninagonists, i.e., MAO inhibitors, e.g., pargyline, synthetic morphineanalogs, e.g., methadone; antiemetics, e.g., metoclopramide; estrogens;and various other serotonin agonists, e.g., tryptophan,5-hydroxytryptophan (5-HTP), fluoxitane, and dexfenfluramine. Moreover,the non-toxic salts of the foregoing prolactin enhancing compoundsformed from pharmaceutically acceptable acids are also useful in thepractice of this invention. Metoclopramide has been found particularlyuseful in the practice of this invention.

Nonlimiting examples of prolactin reducers include prolactin-inhibitingdopamine agonists such as dopamine and certain ergot-relatedprolactin-inhibiting compounds. Nonlimiting examples of dopamineagonists are 2-bromo-alpha-ergo-criptine;6-methyl-8beta-carbobenzyloxy-aminoethyl-10-alpha-ergoline;8-acylaminoergolines, are 6-methyl-8-alpha-(N-acyl)amino-9-ergoline and6-methyl-8alpha-(N-phenylacetyl)amino-9-ergoline; ergocornine;9,10-dihydroergocornine; and D-2-halo-6-alkyl-8-substituted ergolines,e.g., D-2-bromo-6-methyl-8-cyanomethylergoline; carbi-dopa and L-dopa;and lisuride. Moreover, the non-toxic salts of the prolactin-reducercompounds formed with pharmaceutically acceptable acids are also usefulin the practice of this invention. Bromocriptine, or2-bromo-alpha-ergocryptine, has been found particularly useful in thepractice of this invention.

The modulation of immune responses induced by prolactin enhancers orreducers is expected to be dose-dependent over a range of dosages.

In treating mammals, generally, dosages of the prolactin reducer and/orenhancer, respectively, are each given, generally once a day, generallyover a period ranging from about 10 days to about 180 days, buttreatment can continue indefinitely (if necessary or desired) for monthsor even years. The preferred prolactin reducer (accelerated releasebromocriptine) is given daily at dosage levels ranging from about 3micrograms to about 100 micrograms, preferably from about 10 microgramsto about 40 micrograms, per kg of body weight, and the preferredprolactin enhancer (metoclopramide) is given daily at dosage levelsranging from about 5 micrograms to about 200 micrograms, preferably fromabout 5 micrograms to about 100 micrograms, per kg of body weight perday to modify, or alter, the prolactin profile.

Administration of either or both prolactin altering substances can becontinued for a time sufficient to reset the circadian plasma prolactinrhythm to the phase and amplitude modified by administration of theprolactin altering substance, at which time treatment may bediscontinued. If the subject suffers a relapse, treatment may beresumed. The time needed for resetting varies but is generally withinthe range of 30-180 days.

In treating humans, in particular, the prolactin reducer (acceleratedrelease bromocriptine) is generally given at daily dosage levels rangingfrom about 3 micrograms to about 100 micrograms, preferably from about10 micrograms to about 40 micrograms, per kg of body weight (typically0.2-1.5 mg/person/day; preferably 0.8-8 mg). The prolactin enhancermetoclopramide is generally given at daily dosage levels ranging fromabout 1 micrograms to about 50 micrograms, preferably from about 5micrograms to about 20 micrograms, per kg of body weight per day. (Perperson daily dosages range of metoclopramide are typically 0.5 to 5.0mg; preferably 0.5 to 2.0 mg.) Such treatment (using one or both typesof prolactin altering substances) is typically continued over a periodof time ranging from about 10 days to usually about 180 days, resultingin modification and resetting of the immune functions of the patient tothat of a lean, young, healthy person, at which time treatment may bediscontinued. For some patients (e.g. patients in particularly poorphysical condition, or those of an advanced age) it may not be possibleto reset their prolactin rhythm within the above time periods and suchpatients may require a longer, or even continuous, treatment withprolactin enhancers and/or reducers. The dosage and timing informationset forth above is designed for bromocriptine and metoclopramide andwill have to be altered for other agents using the dosage and timingmethodology disclosed herein.

In the practice of this invention, a prolactin reducing compound, and/ora prolactin enhancer are administered daily to a subject preferablyorally, or by subcutaneous, intravenous or intramuscular injection.Dermal delivery systems e.g., skin patches, as well as suppositories andother well-known systems for administration of pharmaceutical agents canalso be employed. Treatment generally lasts between about 10 and about180 days on average in humans. The administration of the prolactinreducer and/or prolactin enhancer in this manner will thus reset thephase and amplitude of the neural oscillators that control the immunesystem to rectify or ameliorate immune function on a long term basis(e.g., several months or years). An improvement or amelioration inimmune function can be assessed by observation of partial or totalrestoration of the ability to mount immune response as described abovein connection with the definition of immune dysfunction. In the case ofautoimmune disease, an improvement or amelioration can best be assessedby a significant reduction or disappearance of a clinical symptomassociated with inflammation caused by the autoimmune disease, forexample: joint pain or swelling or stiffness in rheumatoid arthritis;number of major attacks in chronic-relapsing multiple sclerosis;stabilization or improvement of motor function in chronic progressivemultiple sclerosis; intestinal inflammation in the case of Chron'sdisease; and serological measurements (such as antibody todouble-stranded DNA, complement components and circulating immunecomplexes), number and severity of skin flareups or myalgras,arthralgia, leukopenia, or thrombocytopenia for systemic lupuserythematosus. The symptoms which can be used to monitor efficacy of aregimen in autoimmune disease are generally well-known in the art.

Improvement in ability to mount an immune response against infection canalso be measured by testing for the infectious agent.

The following more specific guidelines will generally be followed toinitially determine bromocriptine administration timing, for a period oftreatment of approximately 26 weeks:

a) Week 1 to Week 6.

First Dosage: If any one of a patient's 07:00, 08:00, 16:00 or 19:00prolactin levels is equal to or higher than 5.0 ng/ml for males or 7.0ng/ml for females, then 0.8 mg of accelerated release bromocriptine isadministered at 06:00 daily.

Second Dosage: Beginning in week 3, a second dosage containing 0.8 mg ofaccelerated release bromocriptine is also administered at 10:30 daily.

b) Week 7 to Week 12.

First dosage: If any one of the 07:00, 08:00, 16:00, or 19:00 prolactinvalues is still equal to or higher than 5.0 ng/ml for males or 7.0 ng/mlfor females, then 1.6 mg of accelerated release bromocriptine areadministered at 06:00. Otherwise, 0.8 mg of accelerated releasebromocriptine is administered at 06:00 daily.

Second Dosage: In addition, if the 19:00 prolactin level is less than orequal to 1.5 ng/ml for males or females then the second dosage of 0.8 mgof accelerated release bromocriptine is administered at 08:30 dailyinstead of at 10:30. If the 19:00 prolactin level is higher than 1.5ng/ml for males and females, then the second dosage continues toadministered at 10:30 daily.

If the 19:00 prolactin level is less than 1.0 ng/ml for males andfemales, then there is no administration of second dosage.

c) Week 13 to Week 26. For both first and second dosages the rules arethe same set forth for Weeks 7-12, subject to the following:

(i) If either the 16:00 or 19:00 prolactin level is equal to or higherthan 5.0 ng/ml for males or 7.0 ng/ml for females, then add anadditional 0.8 mg of accelerated release bromocriptine to the firstdosage, unless the patient is already receiving 2.4 mg of bromocriptinein total. In that case, add the additional 0.8 mg of accelerated releasebromocriptine to the second dosage;

(ii) If the 19:00 prolactin level is lower than 1.5 ng/ml for males orfemales, then the second dosage time is adjusted by administering it 2hours earlier; and

(iii) If each of the 08:00, 16:00 and 19:00 prolactin levels is lessthan 1.0 ng/ml for males or females, then subtract 0.8 mg of acceleratedrelease bromocriptine from the second dosage, or, if there is no seconddosage, then subtract 0.8 mg of accelerated release bromocriptine fromthe first dosage. In the vast majority of patients, the first dosagemust contain a minimum of 0.8 mg of accelerated release bromocriptine.

The time and amount schedules given above are intended as guidelines forbromocriptine administration and those skilled in the art can furtheradjust the precise timing and amount of bromocriptine administrationbased on the actual prolactin profile or key prolactin levels of apatient to be treated. For example, if a patient does not respond (ordoes not respond adequately) to a given dosage or dosages (e.g. 0.8 mg)it (or they) can be increased (e.g. to 1.6 mg).

When needed, metoclopramide (generally daily dosage range is 0.5-5.0mg/person; preferred daily dosage range is 0.5-2.0 mg/person) can beadministered once about one hour before bedtime.

Of course, the foregoing dosages are subject to optimization and it isexpected that there will be minimum and maximum effective dosages. Inother words, adjustment of the prolactin rhythm or levels to regulateimmune response will occur within a specific dosage range. (This is alsoillustrated in Example 2 below for downregulation of immune responsesusing bromocriptine as the prolactin modulator.)

The aspect of the invention directed to a modulation of the immunesystem by resetting the prolactin level profile of a vertebrate subject(animal or human) having an aberrant prolactin profile to conform to orapproach the prolactin profiles for young healthy members of the samespecies and sex (e.g. the baselines of FIGS. 12 et seq.) involvesadministration of a prolactin reducers, or a prolactin enhancer, orboth, at predetermined dosages and times dictated by the aberrant(pre-treatment) prolactin profile of the subject to be treated. Theamounts of prolactin reducers and/or enhancers that are required tobring about this modification are within the same ranges as set forthabove, but the time(s) of administration of these prolactin modulator(s)is determined by reference to how much and when the aberrant profilediffers from the normal prolactin profile (baseline curve). Methods fordetermining the amounts and timing of administration are also set forthin our copending U.S. patent application Ser. No. 07/995,292 and itsC-I-P, Ser. No. 08/264,558, filed Jun. 23, 1994, both incorporated byreference. A preferred accelerated release bromocriptine dosage form hasbeen disclosed in our copending U.S. patent application Ser. No.08/171,897 also incorporated by reference.

(b) Augmenting Immune Responses

As illustrated in Examples 1-5, the present invention provides a methodfor augmenting immune responses (e.g. increased T-cell response orB-cell response etc as described above in connection with the definitionof immune dysfunctions) to increase a subject's ability to fightinfection. This can be accomplished by administration of prolactin oranother prolactin enhancer at a predetermined time during a 24-hourperiod at which increased bloodstream levels of prolactin enhance immuneresponse.

In mice, prolactin injections or administration of prolactin enhancerswere shown to be immunostimulatory during the interval of 4-12 HALOduring which time the immune system responds positively to increasedprolactin levels.

In treating any mammal having a prolactin daily rhythm in accordancewith this aspect of the method of the present invention, the appropriateinterval of positive immunoresponsiveness to increased prolactin mustfirst be ascertained. This can be accomplished by experiment similar tothose of Examples 1-5. Instead of MLR or DTH measurements, well-knownlymphocyte proliferation or lymphocyte activation assays or lymphocytecharacterization methods can be used to assess the effect of increasedprolactin. Once a time point within the appropriate time interval hasbeen identified, administration of the prolactin enhancer can beundertaken. The time of administration can be further optimized byrepeating experiments such as those of Examples 1-5 at time pointsspaced apart from (e.g. within 3 hours of) a time point where prolactinenhancement has been found to be effective in augmenting immuneresponse.

Ascertaining the effective dosage range as well as the optimum amount iswell within the skill in the art. For example, dosages for mammals canbe determined by beginning with a relatively low dose (e.g., 0.8 mgbromocriptine or 0.5 mg of metoclopramide), progressively increasing it(e.g. logarithmically) and assessing the immune responses of the mammalaccording to well-known methods, as detailed in Examples 1-5, below. Theoptimum dosage will be the one generating the maximum or minimum MLR,DTH response, thymic cell count or other measurement of immuneresponsiveness. An effective dosage range will be one that causes atleast a statistically significant alteration of at least one measurementof immune response.

For mammals, generally the amount of prolactin enhancer to augmentimmune response will be within the range of

1 to 50 μg/kg/day

If the enhancer is prolactin, the range will be

10 to 1000 ng/kg/day

For humans, the amounts of prolactin will generally be the same asabove; those for domperidone will be 0.17 to 17 mg/kg/day; 5HTP, 1 to 50mg/kg/day.

Without being bound by theory, it is hypothesized that dailyadministration of exogenous prolactin or increase of endogenousprolactin levels mediates a coordinated cellular preactivation statewhich readies cells for immune responsiveness. Prolactin stimulation oflymphocytes induces the activation of ornithine decarboxylase, nuclearprotein kinase C, IL-2 production, and IL-2 receptor expressionnecessary for enhanced responses to foreign antigen (Gala, R. R., Proc.Soc. Exp. Biol. Med. 198:5-13, 1991; Russel, D. H., Trends Pharm. Sci.10:40-44, 1989). Since prolactin receptors have been identified onpolymorphonucleocytes and macrophages, as well as lymphocytes (Gala, R.R., Proc. Soc. Exp. Biol. Med. 198:5-13, 1991), this "preactivation" mayserve to target various cell activities enhancing immune responses (e.g.MLR and DTH), including the production of thymic hormones known tostimulate MLR (Baxevanis, C. N. et al., Immunopharm 15:73-84, 1988), theproduction of cytokines (Tzehoval, E. et al., Immunopharm. 18:107-113,1989), and enhancement of antigen-presenting ability by increasingexpression of class II MHC (Baxevanis, C. N. et al., J. Immun. 148:1979-1984, 1992) and/or possibly B7 antigens.

Based on previous observations in other physiological systems, the phase(i.e. daily peak) of this immunocellular response rhythm to prolactinmay be entrained directly or centrally by other humoral or neuralfactors. Humoral factors include for example corticosteroid (Meier, A.H., Trans. Am. Fish. Soc. 113:422-431, 1984; Meier, A. H. et al.,Current Ornithology II (ed Johnston R. E.) 303-343, 1984; Cincotta, A.H. et al., J. Endocrinol. 120:385-391, 1989). Neural factors include forexample dopamine (Emata, A. C. et al., J. Exp. Zool. 233:29-34, 1985;Cincotta, A. H. et al., Chronobiol. Int. (in press); Miller, L. J. etal., J. Interdisc. Cycles Res. 14:85-94, 1983). It should be clarifiedthat the daily variation of immunologic responsiveness to prolactin isdistinct from the well-established circadian rhythm of immune activity(Fernandez, J. in Biologic Rhythms in Clinical and Laboratory Medicine(eds. Y. Touitou & E. Haus) 493-503, 1992).

The present invention may be better understood by experiments describedin the Examples below. These Examples are to be considered illustrativeonly of the principles of the invention. Further, since numerousmodifications and changes will readily occur to those skilled in theart, it is not desired to limit the invention to the exact constructionand operation shown and described. Accordingly, all suitablemodifications and equivalents may be used and will fall within the scopeof the invention and the appended claims.

EXAMPLE 1 TIME OF DAY DEPENDENT EFFECTS OF PROLACTIN ON THE ONE-WAYMIXED LYMPHOCYTE REACTION

Groups (n=3-6) of adult male BALB/c and C57BL/6 mice (Charles River,Wilmington, Mass.) were maintained from birth on 12 hour dailyphotoperiods. Ovine prolactin available from Sigma Chemical Co., St.Louis, Mo.) was injected intraperitoneally (1 mg/kg body weight, 20μg/animal/day for 10 days) at 0/24, 4, 8, 12, 16 or 20 HALO. A controlgroup remained untreated. Individual spleen cells (responder cells) werethen obtained from control or experimental mice by standard methods,erythrocytes lysed, and the splenocytes were resuspended in RPMI 1640(Gibco, Grand Island, N.Y.) supplemented with 1 mM L-glutamine 1%penicillin/streptomycin, 0.01 M HEPES, and 1% heat-inactivated normalmouse serum. Stimulator spleen cells were obtained from normal maleC57BL/6 mice, irradiated with 4000 rad of gamma irradiation, washed withHank's balanced salt solution, and resuspended in culture media. 5×10⁵responder cells were added to 5×10⁵ stimulator cells or media alone in atotal volume of 0.2 ml in 96 well flat-bottomed plates. After 96 hr,cell proliferation was assayed by incubation with 1 μCi of ³ H-thymidine(New England Nuclear, Boston, Mass.) and, after an additional 18 hours,cells were harvested and counted in a scintillation counter. Cellsuspensions from each animal were assayed in sextuplicate and expressedas the mean +/- SEM of 3-6 mice per group. FIG. 2 shows a representativeexperiment of three separate experiments.

As can be seen by reference to FIG. 3, prolactin injections made 4-12HALO substantially increased (114%, p<0.05) MLR response toalloantigens. Also increased (to a lesser albeit still significantextent) was the proliferation of nonstimulated responder splenocytesfrom treated animals as compared to the negative controls. It should benoted that injections made 16-20 HALO had no significant effect on MLRresponse. Additionally, injections at light onset (0/24 HALO) resultedin a 66% inhibition of MLR compared to controls.

Thus, the experiment of this example illustrates dramatically theimportance of timing of increases in prolactin level. Increasing theamount of circulating prolactin at different times causes augmentationof immune response to alloantigen or suppression of immune response toalloantigen or produces no significant effect.

The foregoing results have been repeated in another similar experimentthe results of which are shown in FIG. 2 (n=5).

EXAMPLE 2 TIME OF DAY DEPENDENT EFFECTS ON BROMOCRIPTINE ONHAPTEN-SPECIFIC DELAYED-TYPE HYPERSENSITIVITY RESPONSES

Adult male BALB/c mice (5-6 mice per group) maintained on 12 hour dailyphotoperiods were injected daily for 12 days with bromocriptine at 0.5,1.5, 2.5, or 5.0 mg/kg body weight at either 0 or 10 HALO. A controlgroup remained untreated. Six days after the initiation of drugtreatment, treated and positive control (sensitized but nobromocriptine) mice were sensitized to azobenzene arsonate (ABA) bysubcutaneous injection of 3.0×10⁷ ABA-coupled syngeneic male spleencells (Bach, B. A. et al., J. Immunol. 121:1460-1468, 1978). A negativecontrol group remained unsensitized. Six days following sensitization,all mice were challenged in the foot pad with 30 μl of 10 mM ABAsolution. Footpads were measured 24 hours later and the swellingresponse was determined by subtracting the thickness of the non-injectedfootpad from that of the injected footpad. FIG. 10 represents the meanpercent inhibition of footpad swelling compared to the positive controlsobtained from 4 experiments.

As can be seen in FIG. 10, different amounts of bromocriptine produceddifferent effects on the immune system depending on the time of theiradministration. Thus, at 0 HALO, 0.5 mg/kg or 1.5 mg/kg or 2.5 mg/kg ofbromocriptine had no significant effect in inhibiting footpad swelling.5.0 mg/kg of bromocriptine administered 0 HALO produced significantinhibition of DTH responses (i.e. had a significant immunosuppressiveeffect).

On the other hand, at 10 HALO, dosages of 1.5, 2.5 and 5.0 bromocriptinehad a significant suppressive effect. This indicates that the DTHinhibitory (i.e. immunosuppressive) effect of bromocriptine whenbromocriptine is given at 10 HALO is much greater than if given at 0HALO. Bromocriptine inhibits prolactin secretion in mice for about 4-6hours when administered at 1.5 mg/kg and for about 16 hours whenadministered at 5 mg/kg. Thus, the 5.0 mg/kg dosage at 0 HALO produced along-lasting suppression of endogenous prolactin that most likelycarried over to the window of immunoresponsiveness to prolactin. Theseresults show that the dosage of prolactin reducer should not be so highas to ablate the daily prolactin level cycle of the treated mammal butshould be kept at levels that reduce prolactin substantially only duringthe desired interval of day. The results of this Example 2 also showthat the immune responsiveness to prolactin obeys a daily rhythm. Theexperiment of this Example 2 also provides a method for determining theappropriate dosage or dosage range for a prolactin modulator.

The same type of experiment was conducted with prolactin administered at20 μg per animal per day for 12 days at 0 HALO or at 11 HALO. The DTHresponse (expressed as foot pad swelling mm) is shown in FIG. 9 comparedto negative and positive control. The asterisk denotes a significantdifference from positive control.

The foregoing DTH experiments validate the usefulness of the presentinvention in augmenting and suppressing immune responses, includingimmune responses to alloantigen (e.g., allograft rejection).

EXAMPLE 3

The MLR experiment of Example 1 was repeated but bromocriptine (200μg/animal/day or 50 μg/animal/day) was administered for 7 days at 0 and9 HALO. The results are shown in FIG. 6 (A and B). Bromocriptine(prolactin reduction) was found to have no effect on MLR at 0 HALO butwas significantly inhibiting at 9 HALO.

Bromocriptine (50 μg/animal/day for 10 days) was also found to besignificantly more inhibitory of both T-cell and B-cell proliferativeresponses to mitogenic stimulation with either concanavalin A (ConA) inthe culture medium (100% inhibition; p<0.01) (FIG. 7) orlipopolysaccharide (47% inhibition; p<0.01) (FIG. 8) when bromocriptinewas administered at 10 HALO as compared to administration of the sameamount of bromocriptine at 0 HALO in MLR experiments similar to those ofEx. 1. This supports the existence of a daily rhythm of immuneresponsiveness to prolactin.

EXAMPLE 4 TIME OF DAY DEPENDENT EFFECT OF PROLACTIN ENHANCERS ON MLR

The experiment of Example 1 was repeated but the prolactin enhancerdomperidone (which does not cross the blood-brain barrier) wasadministered to mice (n=5 per group) at 23 at 10 HALO to mice in anamount of 1.7 mg/kg/day for seven days. The results, shown in FIG. 4 arethat domperidone significantly increased MLR when administered at 10HALO but not at 23 HALO. The same experiment was conducted with5-hydroxytryptophane (5HTP) in an amount of 25 mg/kg/day for seven days.Again 5HTP did not change MLR when administered at 0 HALO butsignificantly increased MLR when administered at 9 HALO. The results arein FIG. 5. These experiments show that prolactin increase can beachieved indirectly by administration of substances that raisecirculating (blood) prolactin levels.

EXAMPLE 5 TIME OF DAY DEPENDENT EFFECTS OF PROLACTIN ON THYMUS CELLNUMBER

Adult (5 week old) male BALB/c mice (8-10 animals/group) maintained on12 hour daily photoperiods were injected daily for 28 days with ovineprolactin (2.25 mg/kg) at 0 or 11 HALO. A control group remaineduntreated. On day 29 thymuses were removed, cell suspensions wereobtained by mechanical dissociation, and total cell number wasdetermined by counting in a hemocytometer chamber. The results of FIG.11 represent the mean cell number +/- SEM of 8-10 mice per group.

As can be seen in reference to FIG. 11, prolactin treatment at 11 HALOsignificantly increased to 42% the thymus cell number relative tocontrols (p<0.01) whereas prolactin injections at light onset did not.These results indicate that the stimulatory effect of prolactin on theimmune system extends to thymic cells. Additionally, these findings alsosupport that immune responsiveness obeys a circadian rhythm.

In the following Examples 6-10, patients with various autoimmunediseases have been treated with bromocriptine to normalize (or makecloser to normal) and reset their daily prolactin profiles. As a result,the immune function of these individuals improved, in that at least onesymptom due to inflammation associated with the autoimmune diseases thatafflicted each individual was measurably reduced, and/or medication wasreduced or discontinued.

EXAMPLE 6 CROHN'S DISEASE

The subject (male; 20 yrs) was diagnosed with Crohn's disease in 1992based on exploratory surgery and barium X-ray. Approximately 12 inchesof the small intestine were inflamed. The subject received prednisone 40mg/day tapered to zero over a 16 week period.

The subject's 24-hour pre-therapy prolactin profile (generated about 5months after he stopped taking prednisone) is shown graphically as theline labelled "pre-therapy" in FIG. 12. It shows prolactin levels thatare too high throughout the daytime. The subject was given 1.25 mg ofbromocriptine at 08:30 h each day for 20 weeks. A reevaluation profilewas generated for this subject after 20 weeks of treatment and isgraphically shown as the line labelled "Visit 2" in FIG. 12. (Already atVisit 2, the area under the daytime prolactin curve was substantiallyreduced which shows progress but prolactin remained too high from10:00-13:00 and from 16:00-22:00. Ablation of the undesirable earlymorning peak was also observed.) From this time, the dosage wasincreased to 2.5 mg per day at 08:30 h to achieve lower prolactin levelsduring the day. The effects of this change in dosage upon the furtherprolactin profile of the patient (generated 10 months after thecommencement of the 2.5 mg administration) are shown in the linelabelled Visit 3 in FIG. 1, which shows that the daytime male prolactinlevels of the subject were between 2 and 7 ng/ml for most of the daytimeperiod (07:00-22:00) and its prolactin profile has approached thestandard profile in the daytime.

At 15 months from commencement of therapy, the subject still did nothave a proper night time peak although daytime prolactin levels remainedclearly improved. Bromocriptine therapy was continued at 2.5 mg/day fora further 24 weeks (total therapy 20 months).

The clinical improvements to this patient included: (1) avoidance forsurgical resection within this time period (3 yrs. ); (2) no increase ininflamed area of intestine despite discontinuance of prednisone for 2years, based on a comparison of X-rays from first diagnosis with mostrecent (post-therapy); (3) during the time from first diagnosis to endof treatment scarring was minimal as determined by intestinal responseto prednisone treatment; and (4) the patient reported no majorintestinal discomfort during bromocriptine treatment despite no majordietary changes from pre-diagnosis.

EXAMPLE 7 RHEUMATOID ARTHRITIS

The subject (female; 55 yrs old; 5 ft 2 in.; 171.25 lbs) presented with:

(a) rheumatoid arthritis diagnosed in 1972; bursitis in the neck wasdiagnosed in 1992; symptoms included degeneration of the bones in thefingers; medication: 1800 mg of ibuprofen daily (since October 1992)reduced to 400 mg of ibuprofen (ADVIL) twice daily during bromocriptinetreatment and discontinued entirely after 12 weeks of treatment.

(b) obesity: 136% IBW (based on the standard table of Metropolitan LifeInsurance Co. NY, N.Y. available from the company)

The subject's 24 hour pre-therapy prolactin profile is shown graphicallyin FIG. 13 as the lined labelled "OB". The subject's prolactin level wastoo high throughout the day, particularly at 07:00 h. In addition, thenight time peak was shifted forward. The subject was given 1.6 mg ofbromocriptine at 09:00 for the first two weeks and for the followingfour weeks, the subject was given 0.8 mg of bromocriptine at 05:00 and1.6 mg of bromocriptine at 09:00. For the next four weeks (weeks 6-10 ofthe study), the time of the dosage of 1.6 mg of bromocriptine waschanged from 09:00 hr to 10:00 hr. Re-evaluation profiles were generatedfor this patient after 2, 6 and 10 weeks.

The improvements observed in the prolactin profile of this patient aftertwo weeks consisted of prolactin levels throughout the afternoon andearly evening that were normalized or very close to normal. However, theprolactin level was still too high at 07:00. The patient's total dosagewas increased beginning with week 3, to include 0.8 mg of bromocriptineat 05:00 hr, in an attempt to lower the patient's prolactin level at07:00 h. Indeed, the patient's prolactin level at 07:00 hr was reducedto near normal after six weeks of treatment. Therapy lasted 18 weeks. Ascan also be seen in FIG. 13, after 10 weeks of treatment the daytimeprolactin level of the patient remained normal but the night timeprolactin level was reduced below normal levels. Based on substantialclinical experience in prolactin rhythm modifications, however, theinventors believe that a patient afflicted with autoimmune disease whoseprolactin daytime levels have been normalized (or made closer to normal)benefits from the therapy even though night time levels may still be ormay have become abnormal. The present inventors believe that thebenefits to this patient will be further increased when the night timelevels are also normalized.

The clinical improvements in this patient included: cessation of allarthritis medication after week 12 of the treatment and disappearance ofthe following symptoms: swelling, pain and stiffness in the joints; anda loss of body fat of approximately 20 pounds, from 65 pounds to 45pounds. The patient's total weight also dropped over the course of thestudy by 25 lbs. An additional important clinical benefit to thispatient was that the clinical improvements described above have thus farpersisted for 8 months following cessation of the treatment.

EXAMPLE 8 RHEUMATOID ARTHRITIS

The subject (female; 46 yrs old; 5 ft. 5.7 ins; 235 lbs) presented with:

(a) rheumatoid arthritis for approximately six years; the patient wastaking both naproxen (1500 mg) and aspirin (680 mg) daily, as well asibuprofen (200 mg) as needed.

(b) obesity: 156% IBW (based on the standard table of Metropolitan LifeInsurance Co.);

The subject's 24 hour pre-therapy prolactin profile is shown graphicallyin FIG. 14 as the line labeled "Week OB". It shows that pretreatmentprolactin levels (Week O.B.) were too high throughout the day,particularly at 07:00 h. For the first 6 weeks of treatment, the subjectwas given 1.6 mg of bromocriptine at 09:30. From week six through weekten, the subject was given 0.8 mg of bromocriptine at 05:00 hr and 1.6mg of bromocriptine at 10:00 hr. From week 10 through week 18, thesubject was given 1.6 mg of bromocriptine at 05:00 hr and 0.8 mg ofbromocriptine at 10:00 hr. Reevaluation prolactin profiles were taken atseveral intervals, including after 10 and 18 weeks.

The subject's prolactin profile after 18 weeks is shown graphically inFIG. 14. This graph shows that the patient's daytime prolactin levelshave been reduced to normal or near normal throughout most of the day.This graph also shows that the patient lacks a proper night time peak.This patient's profile, however, worsened somewhat after her dosage waschanged from week 10 to week 18 in that a peak appeared at 19:00.

Bromocriptine therapy lasted for a total of 18 weeks.

The clinical improvements in this patient included: discontinuance ofnaproxen (except for one two-week interval during treatment) andsubstitution of tylenol after 18 weeks of treatment, considerablereduction in or disappearance of the following symptoms: pain, jointswelling and stiffness and a loss of body fat of approximately 15pounds. These improvements have thus far persisted for approximatelyfour months after cessation of treatment.

EXAMPLE 9 FIBROMYALGIA

The subject: (female; 38 yrs); presented with fibromyalgia. Symptomsincluded chronic fatigue, stomach disorders and chronic pain in theextremities, including the upper and lower legs. Patient was diagnosedapproximately one year before beginning treatment. There were nopre-treatment medications.

The subject's 24 hour base (pre-therapy) prolactin profile is showngraphically in FIG. 15 as the line labeled "pre-therapy". It shows thatpre-treatment prolactin levels were moderately elevated during the dayand that there was no proper night time peak. Initial dosage ofbromocriptine was 0.625 mg at 6:00 am and metoclopramide was 2.5 mg at10 pm. After four weeks, dosage was changed to 1.25 mg of bromocriptineat 6:00 am and 1.25 mg of metoclopramide at 10 pm. After 8 weeks (4weeks on the modified dosage) the dosage was not further modified. After10 more weeks (total 18 weeks) metoclopramide was discontinued butbromocriptine therapy was continued for a further 4 weeks when it wasdiscontinued as the symptoms had virtually disappeared. Reevaluationprolactin profiles were taken at several intervals, including after 17weeks (visit 3, daytime profile not taken).

The subject's prolactin profile after 4 weeks is shown graphically asthe line labeled "Visit 2" in FIG. 15 and the prolactin profile after 17weeks is shown as the line labelled "Visit 4" in FIG. 15. These graphsshow that the patient's daytime prolactin levels have decreased somewhatat certain points of the day and that the patient has a better nighttime peak.

The clinical improvements in this patient included the disappearance ofthe following symptoms: chronic fatigue, stomach disorders and chronicpain in the extremities, including the upper and lower legs. Theseclinical improvements have persisted for approximately 8 monthsfollowing termination of treatment, which lasted 22 weeks total.

EXAMPLE 10 FIBROMYALGIA

The subject: (female; 27 yrs); presented with fibromyalgia. Symptomsincluded chronic fatigue, stomach disorders, pain and swelling in alljoints, amenorrhea and swelling in the breasts. The patient had beendiagnosed approximately five years before beginning treatment. Patienthad been taking 650 mg of tylenol (daily) and 16 mg of tylenol withcodeine (daily).

The subject's 24-hour base pre-therapy prolactin profile is showngraphically as the line labeled "pre-therapy" in Figure. It shows thatprolactin levels are too high throughout the day, particularly at 13:00hr. For the first 24 weeks of treatment, the patient was administered0.625 mg of bromocriptine at 08:30. For the following 9 weeks oftreatment, the patient was administered 0.625 mg of bromocriptine at05:30 and 0.625 mg of bromocriptine at 09:30. Reevaluation prolactinprofiles were taken at several intervals, including after approximately24 weeks and 35 weeks of treatment.

The subject's prolactin profile after 24 weeks is shown graphically asthe line labeled "24 Weeks" in FIG. 16. This graph shows that thepatient's daytime prolactin levels have been reduced, particularly from10:00 hr to 16:00 hr. The patient's prolactin level is still somewhattoo high in the late afternoon.

The clinical improvements in this patient included: discontinuance ofboth tylenol and tylenol with codeine, and reduction in the followingsymptoms: fatigue, stomach disorders and pain in all joints. Inaddition, a normal menstrual cycle was reinstated and swelling ofbreasts subsided.

What is claimed is:
 1. A method of treating an immune system dysfunctionin a mammal suffering from said dysfunction to at least ameliorate saiddysfunction comprising administering to said mammal:a prolactin reduceronly at a time or times predetermined to reduce the mammal's wakinghours prolactin levels to cause the mammal's daytime prolactin profileto conform to or approach the standard daytime prolactin profile; and aprolactin enhancer only at a time or times predetermined to increase themammal's night time prolactin levels to cause the mammal's night timeprolactin profile to conform to or approach the standard night timeprolactin profile.
 2. The method of claim 1 wherein said prolactinenhancer is selected from the group consisting of metoclopramide,domperidone, 5-hydroxytryptophan, haloperidol, pimozide, phenothiazine,sulpiride, chlorpromazine, serotonin agonists, pargyline, methadone,estrogens, tryptophan, melatonin, fluoxetine, dexfenfluramine, andnon-toxic salts thereof.
 3. The method of claim 1 wherein said prolactinreducer is a member selected from the group consisting of bromocriptine,6-methyl-8-beta-carbobenzy-loxy-aminoethyl-10-alpha-ergoline, an8-acylaminoergoline, ergocornine, 9,10-dihydroergocornine, aD-2-halo-6-alkyl-8-substituted ergoline, carbidopa,L-dihydroxyphenylalanine, lisuride, and pharmaceutically acceptable acidaddition salts thereof.
 4. The method of claim 2 wherein said prolactinenhancer is melatonin, said mammal is a human and said melatonin isadministered at a predetermined time or times between about 19:00 and1:00.
 5. The method of claim 3 wherein said mammal in need of treatmentis a human, said prolactin reducer is bromocriptine, and saidbromocriptine is administered at a predetermined time or times betweenabout 05:00 and 13:00.
 6. A method for treating an autoimmune disease ina mammal suffering from said autoimmune disease comprising administeringto said mammal:a prolactin reducer only at a time or times predeterminedto reduce the patient's daytime prolactin levels to cause the patient'sdaytime prolactin profile to conform to or approach the standard daytimeprolactin profile; and a prolactin enhancer only at a time or timespredetermined to increase the patient's night time prolactin levels tocause the patient's night time prolactin profile to conform to orapproach the standard night time prolactin profile.
 7. The method ofclaim 6 wherein said autoimmune disease comprises rheumatoid arthritis.8. The method of claim 6 wherein said autoimmune disease comprisesCrohns disease.
 9. The method of claim 6 wherein said autoimmune diseasecomprises lupus erythematosus.
 10. The method of claim 7 wherein saidprolactin enhancer is selected from the group consisting ofmetoclopramide, domperidone, 5-hydroxytryptophan, haloperidol, pimozide,phenothiazine, sulpiride, chlorpromazine, serotonin agonists, pargyline,methadone, estrogens, tryptophan, melatonin, fluoxetine,dexfenfluramine, and non-toxic salts thereof.
 11. The method of claim 7wherein said prolactin reducer is a member selected from the groupconsisting of bromocriptine,6-methyl-8-beta-carbobenzy-loxy-aminoethyl-10-alpha-ergoline, an8-acylaminoergoline, ergocornine, 9,10-dihydroergocornine, aD-2-halo-6-alkyl-8-substituted ergoline, carbidopa,L-dihydroxyphenylalanine, lisuride, and pharmaceutically acceptable acidaddition salts thereof.
 12. The method of claim 8 wherein said prolactinenhancer is selected from the group consisting of metoclopramide,domperidone, 5-hydroxytryptophan, haloperidol, pimozide, phenothiazine,sulpiride, chlorpromazine, serotonin agonists, pargyline, methadone,estrogens, tryptophan, melatonin, fluoxetine, dexfenfluramine, andnon-toxic salts thereof.
 13. The method of claim 8 wherein saidprolactin reducer is a member selected from the group consisting ofbromocriptine,6-methyl-8-beta-carbobenzy-loxy-aminoethyl-10-alpha-ergoline, an8-acylaminoergoline, ergocornine, 9,10-dihydroergocornine, aD-2-halo-6-alkyl-8-substituted ergoline, carbidopa,L-dihydroxyphenylalanine, lisuride, and pharmaceutically acceptable acidaddition salts thereof.
 14. The method of claim 12 wherein said mammalis human and said prolactin reducer is bromocriptine administered at apredetermined time or times between about 05:00 and about 13:00.
 15. Themethod of claim 12 wherein said mammal is human and said bromocriptineis administered at a predetermined time or times between about 05:00 andabout 13:00.
 16. The method of claim 14 wherein said mammal is human andsaid prolactin reducer is bromocriptine administered at a predeterminedtime or times between about 05:00 and about 13:00.
 17. The method ofclaim 15 wherein said bromocriptine is administered in an amount in therange of 0.8-8.0 mg/patient/day.
 18. The method of claim 16 wherein saidbromocriptine is administered in an amount in the range of 0.8-8.0mg/patient/day.